514 PHYSIOLOGICAL CHEMISTRY 



boiling for 30 to 60 seconds, longer, provided, however, that the total 

 boiling period, with test tube closed, must not be less than two minutes. 

 Remove the flame and let cool for a little less than two minutes, then 

 add water. Rinse the hot digestion mixture (sometimes turbid from 

 silica) into a 200 c.c. volumetric flask, using for this purpose about 

 125 c c..of water. 



Transfer 10 c.c. of standard ammonium sulphate so!ution containing 

 i mg. of nitrogen into another 200 c.c. volumetric flask. Add i 

 c.c. of the concentrated phosphoric-sulphuric acid mixture, to balance 

 the acid in the unknown, and dilute to a volume of about 150 c.c. 

 When both flasks are thus ready give each flask a whirl and add 30 c.c. 

 of Nessler's reagent. Shake a little more and dilute both flasks to the 

 200 c.c. mark. 



If the unknown Nesslerized digestion mixture is turbid, centrifuge 

 a portion, giving a crystal clear fluid above a white sediment (silica). 

 If the sediment is colored the Nesslerization was not successful and the 

 determination must be discarded. Compare unknown and standard 

 in a colorimeter. 



Reading Standard 

 Calculation.- Reading rf Unknown = mg. of nitrogen m amount of 



urine used. Calculate percentage of nitrogen and daily output in 

 grams. 



Urea 



i. Urease Methods. Principle. These methods depend upon the 

 principle that the enzyme urease is able, at ordinary temperatures, to 

 transform urea, quickly and completely, into ammonium carbonate. 

 Takeuchi 1 in 1909 discovered the presence of this enzyme in the soja 

 or soy bean. The application of this enzyme to the determination 

 of urea in urine, blood, etc., was first proposed by Marshall, 2 whose 

 methods have been modified by Van Slyke and Cullen. 3 These latter 

 investigators prepared a permanent preparation of the enzyme, in a 

 water-soluble form, the use of which makes more convenient the rapid 

 and accurate determination of urea in urine, blood and other biological 

 fluids. 



The urease method is probably the most satisfactory of all methods 

 for the determination of urea. Other nitrogenous constituents such 

 as allantoin are not decomposed by urease. The method involves no 



1 Takeuchi: Journ. Coll. Agr., Tokyo, 1909, Part i. 



2 Marshall, E. K., Jr.: /. Biol. Chem., 14, 283, 1913; 15, 495, 1913; 15, 487, 1913; 17, 

 351, 1914. 



3 Van Slyke D. D., and Cullen, G. E.: /. Am. Med. Ass'n, 62, 1558, 1914. See, also, 

 /. Biol. Chem., 19, 141, 1914. 



